PROJECT SUMMARY The three-dimensional organization of DNA within cells is of profound importance for the appropriate and timely execution of gene expression programs. Recent advances in experimental approaches have allowed the elucidation of the conformation of chromosomes of bacteria and eukaryotes with unprecedented resolution. Bacterial chromosomes are organized into locally folded units and chromosome arms either side of the origin of replication are juxtaposed processively by the action of SMC proteins. In contrast, metazoa have large-scale domains along the linear order of the chromosome, these fall into two distinct types that exclusively associate to form two distinct classes of higher-order compartment. To date, essentially nothing is known about the conformation of chromosomes from organisms in the third domain of life, the Archaea. Many aspects of the information processing (DNA replication, gene transcription) machineries of archaea and eukaryotes are fundamentally related and distinct from those of bacteria. This is despite the prokaryotic nature of archaea, their morphological similarities to bacteria and the presence of simple circular chromosomes, as in many bacteria. Strikingly, preliminary data reveal that the chromosomes of hyperthermophilic archaea of the genus Sulfolobus, like eukaryotes, and distinct from bacteria, possess a large-scale domain architecture with higher-order organization into two distinct classes of compartment. This provocative observation suggests that the fundamental logic of chromosome organization may predate the bifurcation of archaeal and eukaryotic lineages. The preliminary data will be extended to provide a high-resolution 4-D roadmap of the dynamic structure of the archaeal genome. A novel SMC-related protein is implicated in coordinating the structure of the genome and its role will be investigated both in vivo and in vitro using a combination of genetic and biochemical approaches. Finally, the causality of the interplay between the cellular gene expression program and chromosome architecture will be determined. Taken as a whole, this work will exploit a unique and simple model system to elucidate the mechanisms and evolution of the conserved principles governing the higher-order organization of genomes.